The present invention generally relates to systems and methods for culturing cells and more particularly to perfusion operations in a bioreactor. The invention has applications in the culture of animal, insect, and plant cells, for the production of secreted substances such as proteins, antibodies, polypeptides, and viruses. Applications include medical areas such as artificial organs and cell therapy.
Cell culture has generated considerable interest in recent years due to the revolution in genetic engineering and biotechnology. Cells are cultured to make proteins, receptors, vaccines, and antibodies for therapy, research, and for diagnostics.
One limitation to the use of this technology is the high cost of operation. Traditionally, cell culture has been operated in a batch mode. In batch operation, the bioreactor is seeded with a small amount of cells and the cells are grown to high density. The cells secrete the product of interest and eventually die due to lack of nutrients at which point the culture is harvested. This method has several drawbacks-firstly, a large fraction of nutrients are wasted in simply growing up cells and are not used directly for making the product; secondly, product formation is often inhibited due to the buildup of toxic metabolic byproducts; and lastly critical nutrients are often depleted leading to low cell densities and consequently lower product yields.
It has long been recognized that perfusion culture offers better economics. In this operation, cells are retained in the bioreactor, and the product is continuously removed along with toxic metabolic byproducts. Feed, containing nutrients is continually added. This operation is capable of achieving high cell densities and more importantly, the cells can be maintained in a highly productive state for weeks. This achieves much higher yields and reduces the size of the bioreactor necessary. It is also a useful technique for cultivating primary or other slow growing cells. Perfusion operations have tremendous potential for growing the large number of cells needed for human cell and genetic therapy applications.
The central problem in perfusion culture is how to retain the cells in the bioreactor. Prior art can be classified into 3 basic separation technologies xe2x80x941) filtration, 2) gravity sedimentation, and 3) centrifugation. Filtration methods require some means to keep the filter from clogging over the required weeks of operation. Cross-flow filters are typically used. Here a high tangential liquid velocity is used to keep the surface clean. Spinning filters are another embodiment of this concept. Gravity sedimentation can be used to separate the cells and several types of inclined settlers have been reported. The major problem with settlers is the varying sedimentation characteristics of different cells and the difficulty in scale-up to industrial systems. Centrifugation has found limited application in cell culture due to the difficulty in maintaining sterility.
All three current art methods share a common weaknessxe2x80x94in that the liquid from the bioreactor must be pumped through the separation device and the cell-enriched material returned to the bioreactor. Keeping this recirculation loop sterile is difficult, and contamination often occurs. To maintain the high cross-flow velocity necessary to prevent clogging, the cells are subjected to high pumping shear in the recirculation loop and are often damaged. Oxygen depletion can also occur if the pumping rate is too slow. These factors often lead to degradation in product quality and quantity.
From this discussion of prior art, the limitations of current perfusion technology should be clear. As will be apparent in the following discussion, the present invention makes it possible to perform perfusion cell culture without a pump-around loop. This is due to the unique design of the cell retention filter and the rocking motion of the bioreactor.
The present invention solves the problem of filter clogging in perfusion bioreactors by a novel filter design coupled with a bioreactor based on wave-induced agitation. This bioreactor consists of a plastic bag that is partially filled with culture media and inflated to rigidity. The bioreactor is placed on a rocking platform that moves it back and forth through a preset angle and at a preset rocking rate. The rocking motion induces waves in the culture media promoting agitation and oxygen transfer, both essential to good bioreactor performance.
The perfusion filter is constructed such that it is neutrally buoyant with respect to the culture media. It is placed inside the bioreactor in such a manner so that it can move freely with the rocking motion. The bottom surface of the filter consists of a liquid permeable, but cell-retentive membrane. A flexible tube allows the essentially cell-free filtrate to be drawn out from inside the filter. As the bioreactor is rocked, the filter moves rapidly back and forth in the culture media. This back and forth motion serves to clean the filter and allows it to operate without clogging for weeks. Nutrient feed is pumped into the bioreactor and the harvest filtrate is removed continuously, or at periodic intervals.
The present invention provides an inexpensive cell culture bioreactor capable of perfusion operation. It does not require any external pumparound loop or recirculation pump. Thus, it is much simpler, has lower cost, and is less prone to contamination than conventional devices.
The perfusion bioreactor may be used to produce secreted products, produce large amounts of slow growing cells, or function as an artificial organ such as an extracorporeal liver. The simple construction and sterile design make it ideal for hospital use in cell and gene therapy applications.